Cell migration, driven by actin cytoskeletal dynamics, is a fundamentally important cellular behavior. Normal programs of cell migration contribute to organism development and homeostasis, while abnormal migration lays at very heart of pathological processes such as the metastatic spread of malignant tumors. The cAMP-dependent protein kinase (PKA) has oft and long been shown to exert both negative and positive effects on cell migration and cytoskeletal organization. However, little work has been done to reconcile these disparate observations and elucidate the contribution of this venerable and influential kinase to the regulation of cell migration. Preliminary studies show that it is not only the activity of PKA, but also the subcellular distribution of that activity, that is important for chemotactic cell migration. Specifically, PKA regulatory subunits, PKA activity, and the phosphorylation of key cytoskeletal substrates for PKA are significantly enriched in protrusive structures formed at the leading edge of migrating cells. Moreover, both inhibition of PKA activity and disruption of PKA localization inhibit formation of leading edge structures and chemotactic cell migration. This supports an hypothesis in which PKA is activated specifically in the leading edge of migrating cells and this localized activity modulates key regulators of actin cytoskeletal dynamics and cell migration. The proposed work will test this hypothesis by asking two broad questions: How does this localization occur and what are its molecular consequences. A combination of microscopy (3-, 4-, and 5-D imaging; FRET) and biochemical techniques (immunoanalyses, kinase assays, 2-D gel electrophoresis; mass spectrometry) will be used to determine the cellular dynamics of PKA localization to the leading edge and identify the anchoring proteins responsible for it (Specific Aim 1), as well as determine the effects of localized PKA signaling on known cytoskeletal targets for PKA and identify new targets through which PKA might regulate cell motility (Specific Aim 2).